In many individuals HIV-1 broadens from exclusive use of CCR5 as an entry coreceptor (R5 strains) to dual CCR5 & CXCR4 use (R5X4) and, in some, then evolves to variants that primarily use CXCR4 (X4 strains). This transition is important in pathogenesis as it is closely linked to accelerated disease progression, although many important questions remain unanswered including what drives the transition and if it is a cause or consequence of immune decline. Understanding selective coreceptor use is also important because coreceptor blocking agents are being intensively developed as antiretroviral agents. Coreceptor phenotyping is generally carried out in cell lines and current dogma holds that these R5X4 isolates use both CCR5 & CXCR4 for infection in vivo. However, we found that R5X4 dual-tropic strains typically cannot use CCR5 to infect primary lymphocytes (even though lymphocytes are permissive for R5 strains), yet these isolates remain capable of using both CCR5 & CXCR4 in primary macrophages. Thus, many R5X4 dual-tropic variants are "functionally X4" in primary lymphocytes. This selectivity of primary lymphocyte coreceptor use among R5X4 isolates has not been widely recognized, but has major implications for how coreceptor utilization relates to pathogenesis and for the use of coreceptor blocking therapies. Our hypothesis is that that R5X4 strains interact with CCR5 differently from R5 strains leading to differences in lymphocyte CCR5 utilization, and that along with the recognized R5 to R5X4 expansion, viral evolution in vivo is associated with changes in primary cell coreceptor use that are not reflected by phenotyping in cell lines. In particular, we hypothesize that most R5X4 isolates are restricted in their ability to use CCR5 in primary lymphocytes, and that these strains may evolve from early variants that use both pathways in lymphocytes to the majority R5X4 population that is restricted to CXCR4-mediated lymphocyte infection (even though they continue to use CCR5 to infect commonly used cell lines). The goals of this competing renewal application are to better understand pathways used for entry into primary target cells by R5X4 strains, what regulates selective coreceptor use, and what this means for pathogenesis & the use of chemokine receptor antagonists. To do this we will: (1) Define the patterns of CCR5 & CXCR4 utilization for infection of primary cells by dual-tropic HIV-1 subtype B & non-B primary isolates and in relation to viral evolution; (2) Define the interactions between R5X4 gp120 & CCR5. how they differ from R5 gp120. and the implications for coreceptor antagonists; (3) Determine the structural basis for cell- & strain-specific permissive or restricted CCR5 interactions on primary cells, and; (4) Construct SHIV chimeras utilizing naturally-occurring R5 variants we have derived from the HIV-1/89.6 primary isolate swarm. We anticipate that these studies will offer insight into pathways used by primary isolates to enter primary cells relevant to pathogenesis in vivo & entry inhibitor blocking strategies.